All specimens of the new species were collected from the vicinity of Longchuan County and Yingjiang County in western Yunnan, in subtropical evergreen broadleaf forests by searching for flashes or by setting light traps during the night. Specimens are deposited in the following collections: Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China (KIZ-CAS), The Insect Collection of Shanghai Normal University, Shanghai, China (SNUC), collection of Wen-Xuan Bi, Shanghai, China (CBWX), and the collection of Chang-Chin Chen, Tianjin, China (CCCC). Morphological terminology follows Calder (1996) and Costa et al. (2010), except for the wing venation, for which we follow Kukalova-Peck and Lawrence (1993, 2004).

To explore the phylogenetic position of the new Chinese luminescent click beetle, we newly generated 14 mitochondrial genes (13 protein-coding genes and 16S) and two nuclear rRNA genes (18S and 28S) for this species and merge them with the available four-gene Elateridae dataset (18S, 28S, 16S, cox1) by Kundrata et al. (2016, 2018) and Douglas et al. (2018) plus all publicly available Elateridae mitochondrial genomes downloaded from the GenBank (altogether 179 terminals representing 13 subfamilies). Four species of Phengodidae were used as outgroups following Kundrata et al. (2016, 2018) (Suppl. material S1, File 1).

The mitogenome of the new species was obtained from GenBank (accession number MH065615) (He et al. 2019). Because we were not able to obtain high quality reference sequences of 18S and 28S from the Illumina reads previously used for assembling the mitogenome, we assembled 18S and 28S based on another batch of Illumina reads sequenced using fresh specimens collected from type locality later in June 13, 2018, and immediately frozen in liquid nitrogen and stored at -80 °C before use. Total genomic DNA (gDNA) was isolated from two male adults with Sodium Dodecyl Sulfonate method. Library (150-bp insert size) was prepared and sequenced on the Illumina HisSeq4000. Total 38 Gb clean reads were used for assembling 18S and 28S based on the following method: 1) reads were mapped to reference genes of rDNA using mrsFAST v3.3.0 with several iterations (Hach et al. 2014) (first iteration referred to genes downloaded from GenBank (NCBI Resource Coordinators 2016); later iterations referred to assembled contigs in the latest iteration); 2) mapped reads were then assembled using SPAdes v3.11 (Bankevich et al. 2012); 3) tandem contigs were filtered using mereps v2.6 because a huge number of repetitive reads greatly lowers assembly efficiency (Kolpakov et al. 2003); 4) steps one to three were automatically performed for 3–10 iterations until all genes were recovered.

The individual genes were aligned using Mafft online version 7 (https://mafft.cbrc.jp/alignment/server/) (Kuraku et al. 2013) with default parameters. The alignment was displayed and manually curated in Mega7 (Kumar et al. 2016). All 16 aligned genes were concatenated using SequenceMatrix version 1.7.8 (Vaidya et al. 2011). The concatenated matrix was used to calculate the best-fit evolutionary model (GTR+I+G) using PartitionFinder 2.1.1 (Lanfear et al. 2012, 2017). Maximum likelihood (ML) analysis were carried out using RAxML version 7.0.4. (Stamatakis et al. 2008) with 1000 bootstrap replicates. Phylograms were drawn using Interactive Tree of Life (ITOL) (Letunic and Bork 2016).

Our phylogenetic analysis recovered similar topology (Fig. 1) to that of Kundrata et al. (2018), only Tetralobinae were placed in an unsupported terminal clade with Cardiophorinae + Negastriinae, and Dendrometrinae including Diplophoenicus Candèze (Morostomatinae) formed a separate clade. Similar to previously published phylogenies, we obtained low statistical support for the backbone of tree. All subfamilies were monophyletic, except for Dendrometrinae (due to the inclusion of Diplophoenicus) and Lissominae (due to the inclusion of Thylacosterninae). The newly sequenced luminescent species from China was found in an unsupported clade with Oestodinae and Hemiopinae as follows: Oestodes tenuicollis (Randall) + (Hemiops sp. + new luminescent taxon), far from other bioluminescent groups like Thylacosterninae and Agrypninae: Pyrophorini.

Figure 1. 

Inferred phylogenetic position of Sinopyrophorus schimmeli Bi & Li, gen. et sp. nov. within Elateridae based on the concatenated 14 mitochondrial genes (13 protein-coding genes and 16S) and two nuclear ribosomal genes (18S, 28S) using the Maximum Likelihood (ML) analysis. Numbers near each branch indicate ML bootstrap values with 1000 replicates. The same colored shaded areas at the terminals denote the same subfamily. Green bold lines indicate luminescent taxa. The bold red line indicates the presence of luminescent species within the same genus.

He et al. (2019) provided the first preliminary phylogenetic hypothesis for S. schimmeli based on the analysis of 13 protein-coding mitochondrial genes. They found this species sister to a clade containing Dendrometrinae and Elaterinae; however, their sampling of the click-beetle lineages was limited and the position of S. schimmeli was not statistically supported.

Here, we used the most comprehensive dataset of Elateridae to date to elucidate the phylogenetic position of the first known Asian luminescent species. Our analysis (Fig. 1) placed S. schimmeli in a clade with Hemiops and Oestodes, which are both the type genera of the subfamilies Hemiopinae and Oestodinae, respectively, and which were not included in the study by He et al. (2019). However, the relationships among these lineages are not statistically supported and the long branches (see Kundrata et al. 2016) indicate a long-term independent evolution of these groups, which is supported by their distinct morphology. Sinopyrophorinae differ from both Hemiopinae and Oestodinae by having the strongly protruding frontoclypeal region with median longitudinal carina, the antennomeres II and III subequal in length and much shorter than remaining antennomeres (present only in the hemiopine genus Plectrosternus Lacordaire), the posterior margin of pronotum simple, without sublateral incisions or carinae, the prosternal process straight in lateral view, the posterior end of scutellar shield emarginate, the wide mesoventrite with an elongate mesoventral cavity which is only gradually narrowed posteriorly, the bisetose empodium (present in Oestodes, multisetose in Hemiopinae), the abdomen with six or seven ventrites and with a luminous organ, the male genitalia with a median lobe shorter than parameres, and each paramere strongly curved mesad with a dentate inner margin, the female genitalia with a more sclerotized ovipositor, longer paraprocts and styli attached subapically to gonocoxites (apically in Oestodes and Hemiopinae; A. Prosvirov, pers. comm.). These morphological differences in combination with the molecular phylogeny support the subfamilial rank of Sinopyrophorinae, especially when there are no obvious synapomorphies which would define the clade of Sinopyrophorinae + Oestodinae + Hemiopinae, or placement of Sinopyrophorus in any other subfamily. Since the backbone of the Elateridae phylogenetic tree is not or only weakly supported (Kundrata et al. 2016, 2018; this study), future genomic or transcriptomic data might help to resolve the relationships among the deep splits including Sinopyrophorinae.

Our molecular analysis in combination with the morphological investigation confirmed that S. schimmeli does not belong to any of described subfamilies containing bioluminescent species. The clade of Sinopyrophorinae, Oestodinae and Hemiopinae formed one of the basal radiations in Elateridae, far from the Agrypninae: Pyrophorini, which contains the majority of luminescent click beetles (Fig. 1). The position of S. schimmeli far from Pyrophorini is additionally supported by the morphological features such as the pretarsal claws without setae at base (present in Pyrophorini), hind wings with a well-defined wedge cell (absent in Pyrophorini), abdomen with six or seven ventrites (five in Pyrophorini) and the presence of styli on the gonocoxites of ovipositor (styli absent in Pyrophorini). Thylacosterninae, which include the luminescent Balgus schnusei, are regularly recovered inside Lissominae in recent DNA-based analyses (Kundrata et al. 2014, 2016, 2018; Bocak et al. 2018; this study). They differ from Sinopyrophorinae in having the flabellate antennae, deep antennal cavities lying beneath the hypomera, and membranous tarsal lobes (Costa 1984a, Costa et al. 2010). The members of the subfamily Campyloxeninae, which have not been included in any DNA-based study to date, differ from Sinopyrophorinae by much wider frontoclypeal region with a complete frontal carina (frontoclypeal region strongly protruding, relatively narrow and high, longitudinally carinate medially in S. schimmeli), narrowed apical portions of lateral lobes of mesoventrite (wide in S. schimmeli) oval-shaped mesoventral cavity (elongate, gradually narrowed posteriorly in S. schimmeli), abdomen with five ventrites and without a luminous organ (six or seven ventrites, and a large abdominal luminous organ in S. schimmeli), distinctly longer paraprocts, differently shaped aedeagus with the median lobe longer than parameres, and the paramere elongate, with a subapical hook, and apex oriented posteriorly (Costa 1975, Arias-Bohart 2015).

Southern China including Yunnan is a world biodiversity hotspot (Myers et al. 2000) and hosts many endemic beetle species including Elateridae. Intensive entomological research in southern China and neighboring regions is necessary to find out if the newly established Sinopyrophorinae contains more species. Additionally, the discovery of a larva of S. schimmeli may help to better understand the morphological distinctiveness and systematic position of this interesting beetle lineage.

Bioluminescence has evolved independently many times in various organisms (Day et al. 2004). Within Coleoptera, bioluminescence is best known in Lampyridae (fireflies), but also present in Phengodidae, Rhagophthalmidae, and some Elateridae, all within the superfamily Elateroidea (e.g., Oba 2009). Additionally, it was reported also for the larvae of two species belonging to Staphylinidae (Costa et al. 1986, Rosa 2010), which are not closely related to Elateroidea (e.g., Zhang et al. 2018). Within the Elateroidea, bioluminescence independently evolved several times (Bocakova et al. 2007, Sagegami-Oba et al. 2007, Amaral et al. 2014, Fallon et al. 2018) but despite the recent progress in elucidating the phylogenetic relationships within this superfamily (Kundrata et al. 2014), the relationships between the luminescent lineages remain unresolved.

Within Elateridae, nearly all luminescent taxa belong to the agrypnine tribe Pyrophorini, and each of the remaining three smaller groups contain only a single bioluminescent species. All other bioluminescent subfamilies, except Sinopyrophorinae, also include non-luminescent species. Campyloxeninae were placed into Pyrophorini by Stibick (1979) but this was not accepted by later authors (see e.g., Costa et al. 2010). In fact, the members of Campyloxeninae differ diagnostically from Pyrophorini in e.g., the absent setae on tarsal claws, presence of the wedge cell in the hind wing venation and the ovispositor with styli (Costa 1975, Arias-Bohart 2015). Current molecular phylogeny suggests at least three independent origins of bioluminescence within Elateridae, i.e., in Agrypninae: Pyrophorini, Sinopyrophorinae and Thylacosterninae. The presence of a single luminous organ located on abdomen without any prothoracic bioluminescent organs, which is shared by S. schimmeli and the pyrophorine genus Hifo, is therefore an apparent homoplasy. Sequences of the fresh DNA-grade material of Campyloxeninae would help us to better understand the position of this group and if it represents a fourth origin of bioluminescence in Elateridae. All available phylogenetic analyses indicate that the ancestral state of Elateridae was nonluminescent and the luminescence was later obtained in several independent lineages (Bocakova et al. 2007, Sagegami-Oba et al. 2007, Douglas 2011, Kundrata and Bocak 2011, Kundrata et al. 2014).

In Lampyridae, bioluminescence was first gained by larvae as an aposematic warning display, and subsequently gained by adults and co-opted as a sexual signal; the overall trend of courtship is the use of pheromones in ancestral species, then pheromones used in conjunction with photic signals, then the sole use of photic signal (Branham and Wenzel 2001, 2003; Ohba 2004; Stanger-Hall et al. 2018). Males of S. schimmeli have a large luminous organ on abdomen plus very long antennae (longer than in females, hypothesized to be for pheromone detection) and large eyes (larger than in females, and hypothesized to be for bioluminescence detection), which suggest the use of multiple communication channels for mate attraction (Stanger-Hall et al. 2018, and references therein). It is hypothesized that in elaterids, bioluminescence of the abdominal lantern is an optical signal for the intraspecific sexual communication, while the signals from the prothoracic lanterns (if present) serve to warn predators and may also provide illumination in flight (Lall et al. 2000, 2010). Additionally, antennae are usually relatively longer in species which rely greatly on pheromones as sexual signals (Stanger-Hall et al. 2018). Therefore, the external morphology of S. schimmeli suggests the combined sexual communication of both light signals and pheromones. The relatively larger sexual signal sensors of S. schimmeli males in comparison to conspecific females are in agreement with situation in many other insect groups, in which males are the more actively searching sex, with more sensitive sensors (Steinbrecht 1987, Stanger-Hall et al. 2018).

The discovery of S. schimmeli as the first record of a bioluminescent click beetle in Asia shed new light on the geographic distribution and evolution of luminescent click beetles. As a representative of a unique lineage, only distantly related to all other luminescent click beetles, S. schimmeli may serve as a new model taxon in the research of bioluminescence within Coleoptera. A project of de novo genome sequencing of S. schimmeli has already started and should help answer the questions related to the genome characteristics of this taxon (e.g., genome size, heterozygosity etc.), its genomic difference from those of luminescent pyrophorine click beetles, and the genomic basis of the origin of its bioluminescence.